June 20, 1959
DECAPEPTIDE CONTAINING L-LYSINE RESIDUESINSTEAD OF L-ORNITHINE
5,7-Diamino-3-phenyl-v-triazolo(d)pyrimidme(XIV, R = C&,).-Fifteen grams of the 4-anilino-2,5,6-triaminopyrimidine bisulfite (XII) was dissolved in 300 ml. of boiling of glacial acetic water. T~ this solution was added 50 acid, and the solution was stirred while 10 g. of sodium nitrite in 100 ml. of water was slowly added. The solution was then heated on the steam-bath for 1 hr. and cooled. The resulting precipitate was filtered and washed with water.
[CONTRIBUTION FROM
THE
3051
For analysis the compound was recrystallized from N,N-dimethylformamide to yield 4.5 g. of 5,7-diamino-3-phenyl-vtriazolo(d)pyrimidine (XIV, R = C&), ,m.p. >300°. This product was insoluble in aqueous potasslum hydroxide solution. Anal. Calcd. for CioHsN7: 52.5; H, 3.6; N, 43.1. Found: 52.8; H~ 3.9; N~ 43.5. TEMPE,ARIZ.
e,
‘ 9
DEPARTMENT OF MICROBIOLOGY, COLLEGE OF PHYSICIANS AND SURGEONS, COLUMBIA UNIVERSITY]
Synthesis of Peptides Related to Gramicidin S. 1II.I The Decapeptide Containing L-Lysine Residues in Place of L-Omithine2 BY BERNARD F. ERLANGER, WILLIAMV. CURRAN AND NICHOLAS KOKOWSKY RECEIVED DECEMBER 15, 1955 The synthesis of a decapeptide analog of gramicidin S is described. It differs from the latter in being acyclic and containing two L-lysine residues instead of two L-ornithines. I t was obtained in crystalline form as were fourteen of the fifteen polypeptide intermediates, adsorption chromatography being utilized for the purification of the higher intermediates.
This paper reports further progress in the syn- SieberK have recently synthesized gramicidin S, thesis of decapeptide analogs of gramicidin S. utilizing a pentapeptide intermediate described The polypeptide described in this paper is the in paper I‘ of this series. decapeptide H.Val-Lys-Leu-Phe-Pro-Val-Lys-Leu-The synthetic methods used to prepare the decaP~~-P~~.OH-~HC~(L-L-L-D-L)~~(I). peptide I are described in Fig. 2 . As emphasized The structure of gramicidin S and the decapep- in previous papers, choice of synthetic techniques tides synthesized to date are shown in Fig. 1; was governed by the necessity of preventing diagramicidin S is a cyclic decapeptide containing a stereoisomer formation. For this reason, the azide repeated sequence of five amino acid residues. Two route was employed in all cases where acylated of the component amino acid residues, L-ornithine peptides served as intermediates. Fourteen of the and D-phenylalanine, are not frequently en- fifteen compounds were obtained in crystalline countered in naturally occurring polypeptides and form, a positive demonstration of the efficacy of appear rarely, if a t all, in proteins. It is the pur- the azide method for the preparation of complex pose of this program t o ascertain which parts of the polypeptides. chemical structure of gramicidin S are responsible for its antibiotic activity. This objective is being Val-Om-Leu-Phe-Pro-Val-Om-Leu-Phe-Pro pursued by means of the synthesis and antibacterial (L-L-L-D-L)~ Gramicidin S assay of various decapeptide analogs. H .Val-Lys-Leu-Phe-Pro-Val-Lys-Leu-Phe-Pro .OH It was reported earlier4 that decapeptide (I1)l (L-L-L-D-L)Z (I) possessed antibacterial activity, although i t was less potent than gramicidin S. It was proposed H .Val-Om-Leu-Phe.Pro-Val- Om-Leu-Phe-Pro,OH that its lower activity might be the result of its (L-L-L-D-L) (11) greater susceptibility t o bacterial hydrolytic enH .Val-Om-Leu-Tyr-Pro-Val-Orn-Leu-Tyr-Pro. OH zymes because of its acyclic structure. The sug(L-L-L-D-L) (111) gestion was made that the cyclic structure of Fig. 1.-Synthetic peptides. gramicidin S, though not necessary for its antimicrobial activity, prevents destruction of the As in the preparation of decapeptide I11 (ref. peptide by the microorganism. Antibacterial studies of decapeptides I, I1 and 111 and several to be 1, paper 11), it was necessary to perform the synprepared will test this hypothesis and perhaps thesis by the reaction of a tetrapeptide, %Valwith a establish the chemical structure responsible for the ~-TOS.L~S-L~U-P~~.NH.”~(L-L-L-D) hexapeptide, H.Pro-Val-p-Tos.Lys-Leu-Phe-Pro. bactericidal properties of gramicidin S. It should be noted here that Schwyzer and OMe(L-L-L-L-D-L). Decapeptide I1 (ref. 1, paper I) was synthesized by the reaction of two penta( 1 ) Paper I: B. F. Erlanger, H.Sachs and E. Brand, THISJOURpeptide derivatives, but this scheme was not NAL, 76, 1806 (1954); paper 11: B. F. Erlanger, W.V. Curran and feasible here because Z.Val-fi-Tos+Lys-Leu-PheN. Kokowsky, i b i d . , 80, 1128 (1958). (2) This research is supported by the Office of Kava1 Research Pro.NH-NH2(L-L-L-D-L)could not be obtained in under contract N-onr-266(44). A preliminary account appears in the pure crystalline form. Abstracts of the 133rd American Chemical Society meeting, San FranThe pentapeptide, H.Val-p-Tos.Lys-Leu-Phecisco, Calif., ApriI, 1958, p. 27-c. Pro.OMe.HC1 (L-L-L-D-L) (compd. 11) was found (3) For an explanation of the abbreviations, see papers 1 and I1 (ref. 1). Briefly: 2, carbobenzyloxy, COHICH~OCO,p - T o s , p-toluto crystallize in two forms, as needles and as enesulfonyl, CIH~SOI;Leu, leucyl, NH(CHCIH~)CO;Val, valyl, N H rhombohedra, depending upon the quantity of (CHCaHdCO; etc. T h e configurations of the amino acid residues ap. methanol in the recrystallizing solvent. pear in parentheses after the name of the compound. (4) B. F. Erlanger and L. Goode, Nafurc, 174,840 (1951)
( 5 ) R. Schwyzer and P. Sieber, Helo. Chim. Acto, 40, 624 (1957).
3052
Fig 2.
Adsorption chromatography on Florisil was utilized for the purification of the decapeptide Z. (Val-p-Tos.Lys-Leu-Phe-Pro)2.0Me(LL - L-D - ~
) 2
(compd. 14) and its saponified product (compd. IS). The latter was converted to the final product by reduction by sodium in liquid Excess sodium metal was decomposed by the addition of solid Tc'HpC1. After removal of the liquid ammonia the residue was dissolved in water, made acid with hydrochloric acid and then passed through a column of IRlOOA in the acid form. The decapeptide passed through free of cations and evaporation of the eluate resulted in a crystalline product. Recrystallization could be effected by dissolving i t (with warming) in water and then subsequent evaporation of the solvent. The decapeptide was characterized by hydrolysis and by dinitrophenylation studies. Experimental' 1. e-p-Toluenesulfonyl-L-lysine.-Copper hydroxide was prepared by mixing a cold solution of 10.2 g. (0.041 mole) of copper sulfate pentahydrate with 41 ml. (0.082 mole) of cold 2 N sodium hydroxide solution. The gelatinous precipitate was collected by centrifugation, washed several times with cold water and added t o a solution of 12.3 E. (0.067 (6) V. du Vigneaud and 0. K. Behrens, J . B i d . Chem., 117, 27 (1937). ( 7 ) Compounds ace numbered to correspond with numbering in Table I.
mole) of L-lysine monohydrochloride in 34 ml. (0.068 mole) of 2 N sodium hydroxide. T o this solution was added, with vigorous stirring, 17.2 g. (0.090 mole) of p-toluenesulfonyl chloride in 100 ml. of ether followed by a dropwise addition of 40 ml. (0.080 mole) of 2 N sodium hydroxide over a period of 45 minutes. Stirring was continued for an additional 3 hr., after which the chelate was filtered and washed with water, alcohol and ether. T h e light blue product was dissolved in 100 ml. of 2 N hydrochloric acid and treated with hydrogen sulfide t o precipitate the copper as the sulfide. The copper sulfide was removed by filtration, using celite and the filtrate adjusted t o p H 6 with pyridine t o precipitate e-p-toluenesulfonyl-L-lysine, which was collected after standing overnight in the refrigerator; yield 14.0 g. (TOYo). Recrystallization from 5 7 . acetic acid gave 11.9 g. (.%Yo),m.p. 234-237" dec.8 [ L Y ] ~ ~f16.4' D (27, in 6 N hydrochloric acid). Calcd. for C ~ ~ H ~ O (300.4): O~N~S N, 9.4. Found: N , 9..5. 2 . e-p-Toluenesulfonyl-L-lysine Benzyl Ester Hydrochloride.--5.0 g. (0.0167 mole) of e-p-toluenesulfonyl-Llysine (compd. 1) was dissolved in 2 5 ml. of benzyl alcohol containing 7.5 g. of polyphosphoric acid.9 The solution was stirred for 4 hr. in an oil-bath a t 9.5'. After cooling, 165 ml. of anhydrous ether was added and dry hydrogen chloride passed in until the solution was saturated. The precipitated ester hydrochloride, after standing in the refrigerator overnight, was filtered and washed with dry ether; yield 6.48 g. (90.8%), m.p. 172-174'. The crude product was recrystallized from methanol-ether t o give 5.3 g. (7570), [a]=D -7.5 (1% in 0.1 N HC1) (calcd. as free base). A n d . (8) R. Roeske, F. H. C.Stewart, R . J. Stedman and V. du Vigneaud, Tars JOWRXAI.. 7 8 , 5883 (1566), report m.p. 237-238' dec., [ a l Z ' D f13.8 ( c 3, 2 N HC1). (5) Cf.B. F. Erlanger and R . M . Hall, ibid., 76, 5781 (1554).
June 20, 1969
DECAPEPTIDE CONTAINING L-LYSINERESIDUESINSTEAD OF L-ORNITHINE TABLE I PEPTIDE DERIVATIVES
h'0
Compound Z.Val-p-Tos.Lys.OBz(L-L) Z.Val-g-Tos.Lys.OEt(~-~) Z.Val-p-Tos.Lys.NH.NHl(r -L)
4 5 6 7 Z.Val-fi-Tos*Lys-Leu-Phe-Pro.OMe (L-L-L-D-L)
8 H.Leu-Phe.OMe.€ICI(L-D) 9
Z.Val-p-Tos.Lys-Leu-Phe*OMe(L-L-L-D)
10 Z.Vdl-p-Tos. Lys-Leu-Phe.h'H.h'Hn(L-L-L-D)
Mol. wt.
M.p
Calcd. Nitrogen, Found %
3053
Hydrogen, % Carbon, % Calcd. Found Calcd. Found
Mol. formula CrsHeOiKaS CZsHwOlNaS CzsHwOeNiS
624.8 561.7 457.6
124-125.5 139-140 211-211.5
6.9 7.5 12.9
7.0 7.4 12.9
CaHrrOiiNsS Ci6H!rOaNzCl C4xHnOuNrS CuHsrOiNiS
905.1 328.8 $08.0 808.0
219-223.5 217-219 218-220 235-238
9.3 8.5 8.5 12.1
9.2 8.4 8.7 12.1
62.3
62.4
7.1
7.2
62.5 61.0
62.3 61.1
7.1 7.1
7.0 7.0
o c :'
(dec.)
11
H.Val-p-Tos.Lys-Leu-Phe-Pro.OMe.HC1 (L-L-L-D-L)
CaoHssOaNsSCl
807.4
155157
10.4
10.4
58.0
57.9
7.4
7.7
1002.2
157-159
9.8
10.0
62.3
62.0
7.1
7.1
167-169.5
10.8
10.7
58.4
57.9
7.5
7.4
194-196
10.4
10.6
62.0
61.5
7.1
7.2
12 %Pro-Val-p-TosLys-Leu-Phe-l-Pro. OMe (L-L-L-L-D-L)
CiiHiiOiiNrS
13 H.Pro-Val-9-Tos-Lys-Leu-Phe-Pro. OMe. HC1 (L-L-L-L-D-L)
CuHaOiNrSCl
$04.5
14 Z(Val-g-Tos*Lys-Leu-Phe-l-Pro)r.OMe (L-L-L-D-L) I
CoHii~OirNitSt 1644.0
Calcd. for G ~ H z ~ O ~ N (427.0): ~ S C I N, 6.7. Found: N , 6.6. ethyl acetate and washed with cold water, dilute sodium bicarbonate and again with water. The solution was dried 3 . e-p-Toluenesulfonyl-L-lysineEthyl Ester Hydrochloride.--18.7 g. (0.0623 mole) of e-p-toluenesulfonyl-L-lysine over sodium sulfate in the cold and added to a cold-ethyl (compd. 1 ) was suspended in 650 ml. of absolute ethanol acetate solution of H.Leu-Phe-Pro.OMe( L - D - L ) ~ ~previously and cooled in an ice-salt bath. Anhydrous hydrogen chlo- prepared from 7.1 g. (0.0166 mole) of the hydrochloride. ride was passed in until solution was complete (20-30 After standing 18 hr. in the refrigerator and then 24 hr. at minutes). The flask was stoppered and stored in the cold room temperature, the precipitate was filtered off and dried for 2 hr., after which the solvent was removed in vacuo, the in vtcuo to give 11.9 g. (790j0) of a product melting a t 207resulting oil dissolved in 600 ml. of absolute ethanol and re- 211 Recrystallization from aqueous ethanol gave 11.4 treated with hydrogen chloride. The solution, after being g. (76%), m.p. 219-221.5', [CY]*~D -64.5 (0.5% in metha t 4" for 48 hr. was concentrated i n wacuo t o yield a crys- anol). talline solid. Recrystallization from ethanol-ether gave 8. H.Leu-Phe.OMe.HCI( L-D).-~.O g. (0.0107 mole) of 20.4 g. (go%), m.p. 136-137.5", [ a I z 2+~l O . l (2% in 6 N %Leu-Phe.OMe(L-D) (ref. 1, paper I) was dissolved in 150 hydrochloric acid). Calcd. for ClsH2a04N2SCl (364.9): ml. of ethanol containing 5.4 ml. (0.0108 mole) of 2 N h y N, 7.8. Found: N, 7.8. drochloric acid and hydrogenated over activated palladium 4. Z.Val-p-Tos.Lys.OBz(~ - ~ ) . - 1 0 . 3g. (0.041 mole) of until carbon dioxide evolution ceased. The palladium was carbobenzoxy-L-valinelo was dissolved in 50 ml. of dioxane filtered off and the solvents removed in vacuo to yield an oil containing 9.76 ml. (0.41 mole) of tri-n-butylamine and the which crystallized as needles from methanol-ether; yield solution cooled t o 10'; 3.9 ml. (0.041 mole) of ethyl chloro- 2.7 g. (77%), m.p. 217-219' dec., [aIz3D -43.8 (2% in carbonate was added slowly with swirling, after which the methanol). reaction mixture was allowed to stand a t 12' for 30 minutes. 9 . Z~Val-p-Tos.Lys-Leu-Phe.OMe(~-~-~-D).-9.6 g. Then a precooled solution of 17.5 g. (0.041 mole) of a-p- (0 .O 175 mole) of Z.Val-p-Tos. Lys. NH. NH%(L-L) (compd 6) toluenesulfonyl-L-lysine benzyl ester hydrochloride (compd. was dissolved in 80 ml. of 1 Nohydrochloric acid and 295 ml. 2 ) and 9.75 ml. (0.041 mole) of tri-n-butylamine in 50 ml. of of water. After cooling to 0 , 1.25 g. (0.0180 mole) of SOdioxane was added with stirring. The stirring was con- dium nitrite was added and the precipitated azide extracted, tinued for 1 hr., t h e solution left overnight a t room tem- washed and dried in the same manner as described in the perature and then poured into three volumes of water. preparation of Z.Val-p-Tos.Lys-Leu-Phe-Pro.OMe(L-L-LThe precipitated dipeptide derivative was extracted with D-L) (compd. 7). T o the azide solution was added a cold ethyl acetate, the ethyl acetate solution washed with dilute ethyl acetate solution of H.Leu-Phe.OMe( L-D) prepared hydrochloric acid, water, dilute sodium bicarbonate and from 5.75 g. (0.0175 mole) of the hydrochloride (compd. 8). water, and then dried over magnesium sulfate. The com- The solution was allowed to stand overnight in the refrigpound, obtained as an oil by removal of the solvent in wcuo, erator, followed by 4 hr. a t room temperature, after which was crystallized from ethyl acetate-petroleum ether; yield it was cooled again and the product recovered by filtration. 18.8 g. (73.5%), [aImD -27.1 (1% in methanol). ~D (1% in methThe yield was 11.1 g. (Slyo),[ C Y ] -32.0 5 . Z.Val-p-Tos-Lys.OEt(L-L).-This compound was preanol). pared in the same manner as the corresponding benzyl ester 10. Z.Val-p-Tos.Lys-Leu-Phe.NH.NHt( L-L-L-D) .-I 1 .o (compd. 4), except that isobutyl chlorocarbonate was used g. (0.0136 mole) of %Val-p-TosSLys-Leu-Phe.OMe(L-L-Lin the preparation of the mixed anhydride. The yield from D). (compd. 9) was suspended in 165 ml. of absolute 20 g. (0.0548 mole) of the e-p-toluenesulfonyl-L-lysineethyl alcohol containing 4.5 ml. (0.090 mole) of hydrazine ester hydrochloride (compd. 3) was 24 g. (84'%), [ a ] % ~ hydrate. The mixture was refluxed for 7 hr. Most of the -22.6 (1% in methanol). ester dissolves after one hour and hydrazide then starts to pre6. ZVal-p-Tos.Lys.NH.NH2( ~ - ~ ) . - 1 8 . 5 g. (0.03 mole) cipitate. After standing overnight in the refrigerator, the of 2.Val-p-Tos.Lys.OBz (compd. 4) was dissolved in 100 precipitate was collected and washed with cold ethanol and ml. of methanol, 3.7 g. (0.074 mole) of hydrazine hydrate ether; yield 8.79 g. (SO%), m.p. 220-224', [ c Y ] ~ ~-31.6' D added and the solution refluxed for 1 hr. The hydrazide (1% in glacial acetic acid). It was recrystallized from dicrystallized out on cooling and after standing overnight methylformamide-water in the refrigerator, it was filtered and washed with cold meth11. H.Val-p.Tos.Lys-Leu-Phe-Pro-OMe.HCI( L-L-L-Danol; yield 14.3 g. (88%), [cr]l0D-18.5 (1% in glacial acetic ~).-8.24 g. (9.1 mmoles) of &Val-p-TosLys-Leu-Pheacid). Pro.OMe(L-L-L-D-L) (compd. 7) was dissolved in 70 ml. of This compound was also prepared from the ethyl ester (compd. 5) by the same procedure. 12.0 g. (0.0213 mole) 80% acetic acidcontaining9.1 ml. (9.1 mmo1es)of 1Nhydrochloric acid. After addition of activated palladium, hydfoof the ethyl ester gave 10.6 g. (92%) of the hydrazide, gen was passed through the solution until carbon dioxide m.p. 210-213". 7. Z.Val-p-Tos.Lys-Leu-Phe-Pro.OMe( L-L-L-D-L).-~.~ g. evolution ceased. The catalyst was filtered off and the sol(0.0166 mole) of Z.Val-p-Tos.Lys.NH.h'Hz(L-L)(compd. 6 ) vents removed in wucuo. The resulting glass-like solid was was dissolved in a solution containing 86 ml. of glacial acetic crystallized by slow cooling of a solution containing 100 ml. of ethyl acetate and 5 ml. of methanol; yield 5.2 g. acid, 19 ml. of 2 N hydrochloric acid and 250 ml. of water. (70%), [CY]=D -46.6' (0.5% in methanol). After cooling to O", 1.2 g. (0.0174 mole) of sodium nitrite was added. The precipitated azide was extracted into cold (11) J. I. Harris and T. S. Work, ibid., 46, 196 (1950); also ref. 2 , (10) R. L. M.Synge, Biochon. J . , 42, 99 (1948). paper I.
.
.
.
3054
BERNARD F. ERLANGER, WILLIAM v. ~ U R R A PAND ~ NIcrrorAr1s KOKOLVSKY
The product was found to be capable of crysta11.7' iLmg as needles or rhombohedra, depending, apparently, upon the quantity of methanol in the solvent.
ITol. 81
and taken to dryness to yield about 500 rng. of iiiatcrial, 1n.p. 179-185O, [ a l Z o D -85 (2'/&in mctlinnolj. This material \vas reclirornatographed 011 12. Z.Pro-Val-p-Tos.Lys-Leu-Phe-Pro.OMe(L-L-L-L-DPreliminary elution with 450 nil. of 0.5 chloroform yielded about 10 mg. of a colore L).-1.96 g. (7.87 mmoles) of Z,Pro,OH(L)'2 was dissolved in 15 ml. of tetrahydrofuran containing 1.8 ml. (7.55 uct. Then elution was carried out using 426 nil. of l ( ' i , nimoles) of tri-n-butylamine. The solution was cooled to methanol in chloroform, 980 ml. of 1.5CG methanol in chloro0' and 0.99 ml. (7.55 mmoles) of isobutyl chlorocarbonate form and 560 nil. of Zw0 methanol in chloroform, yielding : i was added slowly with swirling. The solution was allowed conibined product wit,h 1n.p. 188-192". This product was to stand for 1 hr. a t 0". During this time the free ester crystallized from 70%, ethanol, to yield 325 i n g . , r i 1 . p . 194 was prepared by dissolving 5.3 g. (6.56 mmoles) of H.Val-pI%', [LYIz5D - 110 (25: in methanol). 15. Z.(Val-p-Tos,Lys-Leu-Phe-Pro)..OH( L-L-L-D-L)*.Tos.Lys-Leu-Phe-Pro.OMe.KC1 (compd. 11) in a small amount of water and adding two equivalents of sodium biX solution of 300 mg. (0.18 nimole) of Z,(Val-p-Tos,Lyscarbonate, causing the precipitation of the free ester, which Leu-Phe-Pro)2,0Me(L-L-L-D-L) (conipd. 14) ill 4 nil. of \vas extracted into ethyl acetate. After washing with water methanol containing 0 . 4 nil. of 1.83 1V aOH was allowed to several times, the ethyl acetate solution was evaporated in stand 3.5 lir. a t 37'. Tlir. solution \ ' tlicii acidified anti s dissolved in ethyl concentrated in z a c u o to ail oil which e'acuo and the residue dried by twice dissolving in 25 ml. of dry benzene arid removing the solvent in vacuo. It was acetate. The ethyl acetate solution was washed with water, dried over anhydrous sodium sulfate :tnd conceiithen dissolved in 50 ml. of tetrahydrofuran, cooled to 0" and trated in vacuo to a glass-like solid; yield 210 mg. added to the mixed anhydride. After standing overnight One hundred and fifty mg. of this material \vas c1iroin:~toin the refrigerator the solution m-as concentrated in tlacuo graphed on 9 g. of Florisil (60-100 iiivsli), aiid the frnctioii and added to 5 volumes of water. The precipitated peptide eluted by 25:'-& inethanol iii chloroforiii \ v a s collected m l c l derivative was extracted with ethyl acetate, which was theii washed with dilute hydrochloric acid, dilute sodium bicar- evaporated to dryness; yield 96 ing.; rieut. equiv., c:ilctl. 1629; found, 168.5; [ a j Z s-96.5 ~ ( 0 , ~ 3 c / in ~ : iiiethaiiol I . bonate and water. The solution was dried over magnesium 16. H,Val-Lys-Leu-Phe-Pro-Val-Lys-Leu-Phe-Pro.OH.sulfate and the ethyl acetate replaced by acetone. Water ~~.~ (0.03 inmole) of %.(\'al-Pwas added to the point of turbidity and after three days a t ~ H C ~ ( L - L - L - D - L ) ? . -mg. (coniptl. 15) was room temperature, the compound crystallized as needles; Tos.Lys-Leu-Phe-Pro)y.O?i(I.-L-I,-D-L)? reduced in liq. NH3 by the addition of 30 ing. o f sodium. yield 5.09 g., m.p. 149-156". The product was recrystal(This was the quantity of sodium required to effect a permalized from acetone-water to give 3.9 g. (BO%), m.p. 157nent blue solution). -1small quantity of S H a C l W:LS d d e d I.iO0, [ L Y ] * ~-i6.3 D (0.576 in methanol). to decompose excess sodium :md the KH3 W:L< allowed to 13. H,Pro-Val-p-Tos.Lys-Leu-Phe-Pro.OMeHC1( L-Levaporate off. After being it the desiccntor over pl~osL-L-D-L) .-4.8 g. (4.79 mmoles) of Z,Pro-Val-p-Tos,Lys-LeuPlie-Pro,Oll.Ie(L-L-L-L-D-L) (conipd. 12) was dissolved in 150 phorus pentoxide for 21 hr, th residue W,IS dissolved in :1 HC1 and p a ~ s e dthrougli a ml. of water plus 3 drops of 8 nil. of methanol and 5.5 ml. ( 5 . 5 mmoles) of 1 N hydrocolumn of 2 g. of I R 100 .Iin t h e acid form. .UII,':md N'L-~ cliloric acid and hydrogenated over activated palladiuin tintil one drop of the solution added to several ml. of water were picked up by the coluinii while tile peptitle passet1 produced no turbidity. ..ifter filtering the palladium, the through. The columii W ~ L Sn x h e d with :iii adclitioi~nl5 nil. of water and all ~vashingswere cornbincd and taken to solvents were removed in vacuo. The oily ester hydrochloride was repeatedly dissolved in absolute ethanol which was dryness in a desiccator i n the presence of PzO5. The rrsitlue evaporated in 'L'UCUO. Finally, the product was taken up was redissolved in 1 inl. of water (slight wmning \vas in rncthanol and ether added until the solution became necessary) and allowed to cv,iporate slowly i n a t1esicc:ttor slightly cloudy. Tlie compound cr) lllized after standing over PzOj. Crystals appeared, Tliey were wIxirated froiu overnight a t room temperature. Recrystnllization from the the supernatant after being cliillecl for \ever:rl (ILL),\ i i i tllc. ~ (0.50/;in refrigerator; yield 20 nig. (613(%). same solveiits gave 2.71 g. (&3:;,), [ a ] * "-78.5 The compound w a s characterized as follows. 0.01 N hydrochloric acid). (a) A fraction of a niiIIigrarn was deposited (111 a sheet of 14. Z,(Val-p-Tos.Lys-Leu-Phe-Pro)~OMe(L-r~-I,-D-L)~.Parafilm. One drop of 6 HC1 was added and the coinpound 2.25 g. (2.76 inmoles) of 2.Val-p-Tos.Lys-Leu-Phe.NH.NHsbrought into solution h y mixing with :i stirring rod drawn to ( I.-L-L-D) (compd. 10) was dissolved in 1.6 ml. of 4 N hydrochloric acid, 28 1111. of glacial acetic acid and 10 ml. of water. a fine tip. The drop of solution was drawn u p into a meltThe solution was cooled t o -2", and 0.23 g. (3.31 mmoles) ing point capillary which was then sealed a t both ends and of sodium nitrite dissolved in 10 ml. of water was added kept a t 110" for 18 hr. The contents of the capillary ?'as O a dropwise with efficient stirring. After addition of the so- deposited as a drop on a watch glass and dried in ~ J G C Z L111 desiccator over P205,then redissolved in water and transdium nitrite the solution was stirred for several minutes, and 60 i d . of cold water was added to ensure complete pre- ferred to Lt'hatman $1 filter paper and chromatograph~d, cipitation of the azide. The mixture was extracted with using butanol-acetic acid-water, 4 : 1: 5, as the develo1)ing cold ethyl acetate washed with cold water, dilute sodium bi- solvent. The amino acids were then developed with 0.1',~ carbonate and water and then dried over magnesium sulfate ninhydrin in acetone and determined quantitatively by thv in the cold. T o this was added a cold dry solution of H.- method of Giri.l3 Leucine, phenylalanine, valine, proline quailtiPro-Val-p-'ros.Lys-Leu-Phe-Pro.OMe(L-L-L-L-D-L) pre- and lysine were fii~intiin apl)roxirn:~teIycqui111(11:1r ties. pared from 2.5 g. (2.76 mmoles) of the hydrochloride (compd. (b) Two mg. of drcapeptide was tlitiitropheiiylated by 13) by the same procedure described for the preparation of hyclrolysis in 6 S HCI % . P r o - V a l - p - T o s . L \ ~ s - L e u - P h e - P r o ~ O ~ ~ e ( I . - I , - L - L - D - Lthe ) method of Sanger." Subsequent (coiiipti. 12). After mixing the solution was allowed t o was followed by extractiuii Lvith ether. The ether p11ase evaporated to dryness xiid chromatographed on IVhatstand a t room temperature nvernight. The solution was was man #I paper, using henzene: 1:; acetic arid, 1: 1 1 5 :LS the then kept :tt r o ( m temperature for 24 hr. and the precipitate developing solvent. The resulting D N P amincr Licit1 Iiad collectetl after chilling in the refrigerator for 4 hr.; yield the same R fas authentic DNP-valine (IZr U.6). 5.22 g. (CiO'jh), m . p . 163-179" dec. (sinters at 151'). ReChromatography of the aqueous fraction (after rvaporapented precipitation by the addition of petroleum ether t o an ethyl acetate solution resulted in 3.9 g. of amorphous tion to dryness and redissolving the residue in water) on paper, using butanol-acetic acid-water 4: 1: ,5, revealed the material with a wide melting range. presence of c-DKP-lysine. Treatment with niiihydrin and I .52 g. of the above material was applied as a chloroform quantitative analysis as above13 showed leucine, phenylsolution to a column of 90 g. of Florisil (60-100 mesh) which alanine, valine and proline in ratios o f approximately was wet with chloroform. After a preliminary elution with 1:1:0.5:1 ( ~ 4 ~ 1 0 ~ Lysine ~ ) . W:LS ahsrnt. 1100 ml. of 0.5% methanol in chloroform and 300 ml. of
